Generation of haploid plants and improved plant breeding

ABSTRACT

Methods and compositions for generating haploid organisms are described.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application claims benefit of priority to U.S. Provisional Patent Application No. 61/248,996, filed Oct. 6, 2009, which is incorporated by reference.

BACKGROUND OF THE INVENTION

Although plant breeding programs worldwide have made considerable progress developing new cultivars with improved disease resistances, yields and other, useful traits, breeding as a whole relies on screening numerous plants to identify novel, desirable characteristics. Very large numbers of progeny from crosses often must be grown and evaluated over several years in order to select one or a few plants with a desired combination of traits.

Standard breeding of diploid plants often requires screening and back-crossing of a large number of plants to achieve the desired genotype. One solution to the problem of screening large numbers of progeny has been to produce haploid plants, the chromosomes of which can be doubled using colchicine or other means to achieve instantly homozygous, doubled-haploid plants.

Thus, marked improvements in the economics of breeding can be achieved via doubled haploid production, since selection and other procedural efficiencies can be markedly improved by using true-breeding (homozygous) progenies. With doubled haploid production systems, homozygosity is achieved in one generation. Thus, the breeder can eliminate the numerous cycles of inbreeding necessary by conventional methods to achieve practical levels of homozygosity. Indeed, true homozygosity for all traits is not even achievable by conventional breeding methods.

BRIEF SUMMARY OF THE INVENTION

The present invention provides for new ways for producing haploid organisms.

In some embodiments, the invention provides a transgenic plant comprising a heterologous transgene expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a recombinantly altered CENH3, CENPC, MIS12, NDC80 or NUF2 polypeptide, wherein in the event the recombinantly altered polypeptide is expressed in a first plant having a corresponding inactivated endogenous CENH3, CENPC, MIS12, NDC80 or NUF2 gene and the first plant is crossed to a wildtype plant, at least 0.1% of resulting progeny are haploid.

In some embodiments, one or two alleles of the endogenous CENH3, CENPC, MIS12, NDC80 or NUF2 genomic coding sequence of the plant is inactivated or knocked out. In some embodiments, all alleles of the endogenous CENH3, CENPC, MIS12, NDC80 or NUF2 genomic coding sequence of the plant is inactivated or knocked out. In some embodiments, the plant, when crossed with a wildtype plant, generates at least 0.1% (or, e.g., 0.5, 1, 2, 5, 10, 20% or more) haploid progeny.

In some embodiments, the polypeptide is a recombinantly altered CENH3 polypeptide. In some embodiments, the polypeptide comprises a heterologous amino acid sequence of at least 5 amino acids linked to a protein comprising a CENH3 histone-fold domain, wherein the amino acid sequence is heterologous to the CENH3 histone-fold domain. In some embodiments, the heterologous amino acid sequence is linked directly to the CENH3 histone-fold domain and the polypeptide lacks a CENH3 tail domain. In some embodiments, the heterologous amino acid sequence is linked to the CENH3 histone-fold domain via an intervening protein sequence. In some embodiments, the intervening protein sequence comprises a non-CENH3 histone H3 tail domain. In some embodiments, the non-CENH3 histone H3 tail domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:95, or a fragment thereof at least 20 amino acids long.

In some embodiments, the intervening protein sequence comprises a CENH3 tail domain. In some embodiments, the intervening protein sequence comprises a histone H3 tail domain and a heterologous histone CENH3 tail domain. In some embodiments, the CENH3 tail domain is heterologous to the CENH3 histone-fold domain.

In some embodiments, the heterologous amino acid sequence is at least 10 amino acids long. In some embodiments, the intervening protein sequence comprises a histone H3 tail domain and a heterologous histone CENH3 tail domain. the heterologous amino acid sequence comprises green fluorescent protein. In some embodiments, the heterologous amino acid sequence disrupts centromeres. In some embodiments, the CENH3 histone-fold domain is selected from the group consisting of SEQ ID NOs: 49-94.

In some embodiments, the polypeptide comprises a non-CENH3 tail domain linked to a CENH3 histone-fold domain.

In some embodiments, the polypeptide comprises a CENH3 histone-fold domain and a truncated CENH3 tail domain, wherein the amino terminus of the tail domain is truncated relative to the plant's endogenous tail domain. In some embodiments, the truncated CENH3 tail domain lacks three or more amino terminal amino acids of the endogenous tail domain. In some embodiments, a heterologous amino acid sequence is linked to the amino terminus of the truncated tail domain. In some embodiments, the heterologous amino acid sequence is at least 10 amino acids long. In some embodiments, the heterologous amino acid sequence comprises green fluorescent protein. In some embodiments, the heterologous amino acid sequence disrupts centromeres. In some embodiments, the CENH3 histone-fold domain is selected from the group consisting of SEQ ID NOs: 49-94.

In some embodiments, the polypeptide is a recombinantly altered CENPC, MIS12, NDC80 and NUF2 polypeptide.

The present invention also provides for an isolated nucleic acid comprising a polynucleotide encoding a polypeptide, wherein the polypeptide comprises:

a non-CENH3 tail domain linked to a CENH3 histone-fold domain; or

a truncated CENH3 tail domain linked to a CENH3 histone-fold domain, wherein the amino terminus of the tail domain is truncated.

The present invention also provides for a plant comprising a silenced CENH3 or one or two copies of an allele of a knocked out, inactivated, or mutated endogenous CENH3 gene.

The present invention also provides for method of generating a haploid plant, the method comprising,

crossing a plant expressing an endogenous CENH3 protein to a transgenic plant comprising a heterologous transgene expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a recombinantly altered CENH3, CENPC, MIS12, NDC80 or NUF2 polypeptide, wherein in the event the recombinantly altered polypeptide is expressed in a first plant having a corresponding inactivated endogenous CENH3, CENPC, MIS12, NDC80 or NUF2 gene and the first plant is crossed to a wildtype plant, at least 0.1% of resulting progeny are haploid; and selecting F1 haploid progeny generated from the crossing step.

In some embodiments, the plant expressing an endogenous CENH3 protein is the pollen parent of the cross.

In some embodiments, the plant expressing an endogenous CENH3 protein is the ovule parent of the cross.

In some embodiments, the method further comprises converting at least one selected haploid plant into a doubled haploid plant.

A method of making a transgenic plant comprising a heterologous transgene expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a recombinantly altered CENH3, CENPC, MIS12, NDC80 or NUF2 polypeptide, wherein in the event the recombinantly altered polypeptide is expressed in a first plant having a corresponding inactivated endogenous CENH3, CENPC, MIS12, NDC80 or NUF2 gene and the first plant is crossed to a wildtype plant, at least 0.1% of resulting progeny are haploid, the method comprising,

transforming plant cells with a nucleic acid comprising the expression cassette; and

selecting transformants comprising the nucleic acid, thereby making the plant.

In some embodiments, the present invention provides an isolated polynucleotide encoding a polypeptide, wherein the polypeptide comprises:

an amino acid sequence of at least 5 amino acids linked to a protein comprising a CENH3 histone-fold domain, wherein the amino acid sequence is heterologous to the CENH3 histone-fold domain; or

a protein comprising a CENH3 histone-fold domain and a truncated CENH3 tail domain, wherein the amino terminus of the tail domain is truncated.

In some embodiments, the heterologous amino acid sequence is linked directly to the CENH3 histone-fold domain. In some embodiments, the polypeptide lacks a CENH3 tail domain.

In some embodiments, the heterologous amino acid sequence is linked to the CENH3 histone-fold domain via an intervening protein sequence. In some embodiments, the intervening protein sequence comprises a non-CENH3 histone H3 tail domain. In some embodiments, the intervening protein sequence comprises a CENH3 tail domain. In some embodiments, the CENH3 tail domain is heterologous to the CENH3 histone-fold domain. In some embodiments, the non-CENH3 histone H3 tail domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:95, or a fragment thereof at least 20 amino acids long. In some embodiments, the intervening protein sequence comprises a histone H3 tail domain and a heterologous histone CENH3 tail domain.

In some embodiments, the heterologous amino acid sequence is at least 3, 5, 10, 15, 20, 30, or 50 amino acids long, optionally lacking a fixed secondary structure.

In some embodiments, the heterologous amino acid sequence comprises green fluorescent protein.

In some embodiments, the heterologous amino acid sequence disrupts centromeres.

In some embodiments, the CENH3 histone-fold domain is selected from the group consisting of SEQ ID NOs: 49-94.

In some embodiments, the polypeptide comprises a protein comprising a CENH3 histone-fold domain and a truncated CENH3 tail domain, wherein the amino terminus of the tail domain is truncated.

In some embodiments, the truncated CENH3 tail domain lacks at least 1, 2, 3, 4, 5, 6, 10, 15, or 20 amino terminal amino acids of the endogenous tail domain. In some embodiments, a heterologous amino acid sequence is linked to the amino terminus of the truncated tail domain. In some embodiments, the heterologous amino acid sequence is at least at least 3, 5, 10, 15, 20, 30, or 50 amino acids long. In some embodiments, the heterologous amino acid sequence comprises green fluorescent protein. In some embodiments, the heterologous amino acid sequence disrupts centromeres.

In some embodiments, the CENH3 histone-fold domain is selected from the group consisting of SEQ ID NOs: 49-94.

The present invention also provides an expression cassette comprising any of the above-listed the polynucleotides, wherein the expression cassette comprises a promoter operably linked to the polynucleotide encoding a polypeptide. In some embodiments, the invention provides for a vector comprising the expression cassette.

In some embodiments, the invention provides for a plant comprising the expression cassette.

In some embodiments, the heterologous histone tail domain comprises a histone H3 tail domain or a heterologous histone CENH3 tail domain.

In some embodiments, the polypeptide comprises a histone H3 tail domain and a histone CENH3 tail domain.

In some embodiments, the plant comprises a silenced CENH3 or one or two copies of an allele of a knocked out or mutated endogenous CENH3 gene.

In some embodiments, the expression cassette is integrated into the chromosome of the plant.

The present invention also provides for a plant comprising a silenced CENH3 or one or two copies of an allele of a knocked out or mutated endogenous CENH3 gene.

The present invention also provides for a method of generating a haploid plant. In some embodiments, the method comprises, crossing a plant expressing an endogenous CENH3 protein to the plant as described herein (e.g., expressing a tailswap protein); and selecting F1 haploid progeny generated from the crossing step.

In some embodiments, the plant expressing an endogenous CENH3 protein is the pollen parent of the cross.

In some embodiments, the plant expressing an endogenous CENH3 protein is the ovule parent of the cross.

In some embodiments, the method further comprises converting at least one selected haploid plant into a doubled haploid plant.

Other aspects of the invention will be clear from the remainder of the text herein.

DEFINITIONS

An “endogenous” gene or protein sequence refers to a non-recombinant sequence of an organism as the sequence occurs in the organism before human-induced mutation of the sequence. A “mutated” sequence refers to a human-altered sequence. Examples of human-induced mutation include exposure of an organism to a high dose of chemical, radiological, or insertional mutagen for the purposes of selecting mutants, as well as recombinant alteration of a sequence. Examples of human-induced recombinant alterations can include, e.g., fusions, insertions, deletions, and/or changes to the sequence.

The term “promoter” refers to regions or sequence located upstream and/or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. A plant promoter can be, but does not have to be, a nucleic acid sequence originally isolated from a plant.

The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

The term “plant” includes whole plants, shoot vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers and floral organs/structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e.g., guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.

A polynucleotide or polypeptide sequence is “heterologous to” an organism or a second sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g. a genetically engineered coding sequence or an allele from a different ecotype or variety). In another example, a CENH3 tail domain from a first species is heterologous to a CENH3 histone-fold domain from a second species.

“Recombinant” refers to a human manipulated polynucleotide or a copy or complement of a human manipulated polynucleotide. For instance, a recombinant expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology, Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In another example, a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature. For instance, human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second polynucleotide. One of skill will recognize that polynucleotides can be manipulated in many ways and are not limited to the examples above.

A “transgene” is used as the term is understood in the art and refers to a heterologous nucleic acid introduced into a cell by human molecular manipulation of the cell's genome (e.g., by molecular transformation). Thus a “transgenic plant” is a plant comprising a transgene, i.e., is a genetically-modified plant. The transgenic plant can be the initial plant into which the transgene was introduced as well as progeny thereof whose genome contain the transgene.

The term “corresponding” as used herein is used to mean “respective.” For example, where it is said that a plant contains a recombinantly altered copy of a protein selected from A, B, and C, and the plant also contains a “corresponding” mutated endogenous copy of the gene selected from a gene encoding A, B, or C, if the plant contains a recombinantly altered protein A, the corresponding mutated endogenous copy would also be A. Alternatively, if the plant contains a recombinantly altered protein B, the corresponding mutated endogenous copy would also be B, etc.

The phrase “nucleic acid” or “polynucleotide sequence” refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. Nucleic acids may also include modified nucleotides that permit correct read through by a polymerase, and/or formation of double-stranded duplexes, and do not significantly alter expression of a polypeptide encoded by that nucleic acid.

The phrase “nucleic acid sequence encoding” refers to a nucleic acid which directs the expression of a specific protein or peptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein. The nucleic acid sequences include both the full length nucleic acid sequences as well as non-full length sequences derived from the full length sequences. It should be further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.

The phrase “host cell” refers to a cell from any organism. Exemplary host cells are derived from plants, bacteria, yeast, fungi, insects or other animals. Methods for introducing polynucleotide sequences into various types of host cells are well known in the art.

An “expression cassette” refers to a nucleic acid construct, which when introduced into a host cell (e.g., a plant cell), results in transcription and/or translation of a RNA or polypeptide, respectively. An expression cassette can result in transcription without translation, for example, when an siRNA or other non-protein encoding RNA is transcribed.

Two nucleic acid sequences or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The term “complementary to” is used herein to mean that the sequence is complementary to all or a portion of a reference polynucleotide sequence.

Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977), and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. Software for performing BLAST analyses is publicly available on the Web through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

“Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 25% sequence identity to a designated reference sequence. Alternatively, percent identity can be any integer from 25% to 100%, for example, at least: 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. One of skill will recognize that the percent identity values above can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 40%. Percent identity of polypeptides can be any integer from 40% to 100%, for example, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, polypeptides that are “substantially similar” share sequences as noted above except that residue positions that are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Exemplary conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a sequence alignment of various CENH3 proteins (A. thaliana H3.3=SEQ ID NO:1; Human H3.3=SEQ ID NO:2; C. albicans=SEQ ID NO:106; Human=SEQ ID NO 107; A. thaliana=SEQ ID NO:10; Poplar=SEQ ID NO:11; Rice=SEQ ID NO:108).

DETAILED DESCRIPTION I. Introduction

The present invention is based, in part, on the surprising discovery that elimination of an endogenous CENH3 in combination with expression of a heterologous protein comprising an altered CENH3 results in a plant that has useful properties for breeding. For example, when a plant that lacks an endogenous CENH3 protein, and expresses a protein comprising (listed from amino terminus to carboxyl terminus) a GFP tag, a non-CENH3 tail domain and a CENH3 histone fold domain, is crossed to a plant having an endogenous CENH3 protein, a portion of the resulting progeny lack all chromosomes derived from the parent plant that expresses an altered version of CENH3. Thus, the invention allows for the production of haploid progeny. Haploid plants are useful, for example, for improving and speeding breeding.

CENH3 is a member of the kinetochore complex, the protein structure on chromosomes where spindle fibers attach during cell division. Without intending to limit the scope of the invention, it is believed that the observed results are due in part to generation of a kinetochore protein that acts more weakly than wildtype, thereby resulting in functional kinetochore complexes (for example, in mitosis), but which result in relatively poorly segregating chromosomes during meiosis relative to chromosomes also containing wildtype kinetochore complexes from the other parent. This results in functional kinetochore complexes when the altered protein is the only isoform in the cell, but relatively poorly segregating chromosomes during mitosis when the parent with altered kinetochores is crossed to a parent with wildtype kinetochore complexes. In addition to CENH3, other kinetochore proteins include, e.g., CENPC, MCM21, MIS12, NDC80, and NUF2. Accordingly, the present invention provides for plants, fungi, or animals (or cells thereof) that express a recombinant mutated kinetochore protein (including but not limited to CENH3, CENPC, MCM21, MIS12, NDC80, and NUF2) that disrupts the centromere, and/or plants, fungi, or animals (or cells thereof) in which at least one or both copies of an allele of the endogenous CENH3 gene has been knocked out, mutated to reduce or eliminate its function, or silenced. As explained in more detail below, the mutated kinetochore protein can be mutated in many different ways, including but not limited to, as a “tailswap” protein, comprising a CENH3 histone-fold domain and a heterologous amino terminal sequence. The present invention also provides for methods of generating a haploid plant by crossing a plant expressing a mutated kinetochore protein (including but not limited to a tailswap CENH3 protein), and not expressing an endogenous CENH3 protein, to a plant that expresses an endogenous CENH3 protein.

II. Kinetochore Proteins

A. CENH3 Proteins

CENH3 proteins are a well characterized class of proteins that are variants of H3 histone proteins and that are specialized proteins associated with the centromere. CENH3 proteins are characterized by a variable tail domain, which does not form a rigid secondary structure, and a conserved histone fold domain made up of three α-helical regions connected by loop sections. Additional structural and functional features of CENH3 proteins can be found in, e.g., Cooper et al., Mol Biol Evol. 21(9):1712-8 (2004); Malik et al., Nat Struct Biol. 10(11):882-91 (2003); Black et al., Curr Opin Cell Biol. 20(1):91-100 (2008). CENH3 proteins are one of the proteins that form the kinetochore complex.

A wide variety of CENH3 proteins have been identified. See, e.g., SEQ ID NOs:1-48. It will be appreciated that the above list is not intended to be exhaustive and that additional CENH3 sequences are available from genomic studies or can be identified from genomic databases or by well-known laboratory techniques. For example, where a particular plant or other organism species CENH3 is not readily available from a database, one can identify and clone the organism's CENH3 gene sequence using primers, which are optionally degenerate, based on conserved regions of other known CENH3 proteins.

The practice of the present invention will generally employ conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Gennaro, A. R., ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Hardman, J. G., Limbird, L. E., and Gilman, A. G., eds. (2001) The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill Co.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press, Inc.; Weir, D. M., and Blackwell, C. C., eds. (1986) Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications; Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999) Short Protocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press; Newton, C. R., and Graham, A., eds. (1997) PCR (Introduction to Biotechniques Series), 2nd ed., Springer Verlag.

i. CENH3 Histone Fold Domain

As noted above, the CENH3 histone fold domain is conserved between CENH3 proteins from different species. The CENH3 histone fold domain can be distinguished by three α-helical regions connected by loop sections. While it will be appreciated that the exact location of the histone fold domain will vary in CENH3 proteins from other species, it will be found at the carboxyl terminus of an endogenous (wildtype) CENH3 protein. Thus, in some embodiments, a CENH3 protein can be identified in an endogenous protein as having a carboxyl terminal domain substantially similar (e.g., at least 30%, 40%, 50%, 60%, 70%, 85%, 90%, 95% or more identity) to any of SEQ ID NOs: 49-94. An alignment of a selection of CENH3 proteins is provided in FIG. 1 and illustrates areas of conservation in the histone fold domain.

The border between the tail domain and the histone fold domain of CENH3 proteins is at, within, or near (i.e., within 5, 10, 15, 20, or 25 amino acids from the “P” of) the conserved PGTVAL (SEQ ID NO:114) sequence. The PGTVAL (SEQ ID NO:114) sequence is approximately 81 amino acids from the N terminus of the Arabidopsis CENH3 protein, though the distance from the N terminus of different endogenous CENH3 proteins varies. See, for example, the sequence listing. Thus, in some embodiments, the histone fold region of CENH3 employed in the tailswap proteins includes all of the C-terminal amino acids of an endogenous CENH3 protein (or a protein substantially similar to the endogenous sequence) up to and including the PGTVAL (SEQ ID NO:114). SEQ ID NOS:49-94 reflect this option. In other embodiments, the tailswap proteins of the invention can comprise more or less of the CENH3 sequence. For example, in some embodiments, the tailswap will comprise the C-terminal sequence of a CENH3 protein, but only up to an amino acid 5, 10, 15, 20, or 25 amino acids in the C-terminal direction from the “P” of the conserved PGTVAL (SEQ ID NO:114) sequence. In some embodiments, the tailswap will comprise the C-terminal sequence of a CENH3 protein, but only up to an amino acid 5, 10, 15, 20, or 25 amino acids in the N-terminal direction from the “P” of the conserved PGTVAL (SEQ ID NO:114) sequence.

ii. CENH3 Histone Tail Domain

Although the histone-fold domain of CENH3 evolves more rapidly than that of conventional H3, CENH3 and H3 histone-fold domains can still be aligned. In contrast, N-terminal tail domains of CENH3 are highly variable even between closely related species. Histone tail domains (including CENH3 tail domains) are flexible and unstructured, as shown by their lack of strong electron density in the structure of the nucleosome determined by X-ray crystallography (Luger et al., Nature 389(6648):251-60 (1997)).

iii. Mutated CENH3 Proteins

Any number of mutations of CENH3 can be introduced into a CENH3 protein to generate a mutated (including but not limited to a recombinantly altered) CENH3 protein capable of generating haploid plants when expressed in a plant lacking, or having suppressed expression of, an endogenous CENH3 protein, and where the resulting transgenic plant is crossed to a plant expressing a wildtype CENH3 protein. Active mutated CENH3 proteins can be identified, for example, by random mutagenesis, by single or multiple amino acid targeted mutagenesis, by generation of complete or partial protein domain deletions, by fusion with heterologous amino acid sequences, or by combinations thereof. “Active” mutant CENH3 proteins refer to proteins, which when expressed in a plant in which CENH3 is knocked out or inactivated, results in viable plants, which viable plants when crossed to a wildtype plant, produce haploid progeny at a more than normal frequency (e.g., at least 0.1, 0.5, 1, 5, 10, 20% or more). Mutated CENH3 proteins can be readily tested by recombinant expression of the mutated CENH3 protein in a plant lacking endogenous CENH3 protein, crossing the transgenic plant (as a male or female, depending on fertility) to a plant expression wildtype CENH3 protein, and then screening for the production of haploid progeny.

In some embodiments, the mutated CENH3 protein is identical to an endogenous CENH3 protein but for 1, 2, 3, 4, 5, 6, 7, 8, or more (e.g., 1-2, 1-4, 1-7) amino acids. For example, in some embodiments, the endogenous wildtype protein from the plant is identical or substantially identical to any of SEQ ID NOs: 1-48 and the mutated CENH3 protein differs from the endogenous CENH3 protein by 1, 2, 3, 4, 5, 6, 7, 8, or more (e.g., 1-2, 1-4, 1-7) amino acids.

In some embodiments, the mutated CENH3 protein contains a CENH3 histone-fold domain identical to the CENH3 histone-fold domain of an endogenous CENH3 protein but for 1, 2, 3, 4, 5, 6, 7, 8, or more (e.g., 1-2, 1-4, 1-7) amino acids. For example, in some embodiments, the endogenous wildtype CENH3 histone-fold domain from the plant is identical or substantially identical to any of SEQ ID NOs: 49-94 and the mutated CENH3 protein contains a CENH3 histone-fold domain that differs from the endogenous CENH3 protein histone-fold domain by 1, 2, 3, 4, 5, 6, 7, 8, or more (e.g., 1-2, 1-4, 1-7) amino acids.

It is believed that active CENH3 mutants include, for example, proteins comprising:

a heterologous amino acid sequence (including but not limited to GFP) linked to a CENH3 truncated or complete tail domain or non-CENH3 tail domain, either of which is linked to a CENH3 histone fold domain; or

a CENH3 truncated tail domain, a heterologous CENH3 tail domain, or non-CENH3 tail domain, either of which is linked to a CENH3 histone fold domain.

In some embodiments, the mutated CENH3 protein comprises a fusion of an amino-terminal heterologous amino acid sequence to the histone-fold domain of a CENH3 protein. Generally, the histone fold domain will be identical or at least substantially identical to the CENH3 protein endogenous to the organism in which the mutated CENH3 protein will be expressed. In some embodiments, the mutated CENH3 protein will include a histone tail domain, which can be, for example, a non-CENH3 tail domain, or a CENH3 tail domain.

It is believed that a large number of different amino acid sequences, when linked to a protein comprising a CENH3 histone-fold domain and a sequence that can function as or replace a histone tail domain, can be used according to the present invention. In some embodiments, the heterologous sequence is linked directly to the CENH3 histone-fold domain. In some embodiments, the heterologous sequence is linked is an intervening amino acid sequence to the CENH3 histone-fold domain. In some embodiments, the intervening amino acid sequence is an intact or truncated CENH3 tail domain. In some embodiments, the heterologous amino acid sequence, in combination with the histone-fold domain, will be sufficient to prevent the lethality associated with loss of endogenous CENH3, but will sufficiently disrupt centromeres to allow for production of haploid progeny, as discussed herein. Thus, in some embodiments, the heterologous amino acid sequence will comprise a portion that is, or mimics the function of, a histone tail domain and optionally can also comprise a bulky amino acid sequence that disrupts centromere function. In some embodiments, at least a portion of the heterologous amino acid sequence of the mutated CENH3 protein comprises any amino acid sequence of at least 10, 20, 30, 40, 50, e.g., 10-30, 10-50, 20-50, 30-60 amino acids, optionally lacking a stable secondary structure (e.g., lacking coils, helices, or beta-sheets). In some embodiments, the tail domain has less than 90, 80, or 70% identity with the tail domain (e.g., the N-terminal 135 amino acids) of the CENH3 protein endogenous to the organism in which the mutated CENH3 protein will be expressed. In some embodiments, the tail domain of the mutated CENH3 protein comprises the tail domain of a non-CENH3 histone protein, including but not limited to an H3 histone protein. In some embodiments, the tail domain of the mutated CENH3 protein comprises the tail domain of a non-CENH3 histone protein endogenous to the organism in which the mutated CENH3 protein will be expressed. In some embodiments, the tail domain of the mutated CENH3 protein comprises the tail domain of a homologous or orthologous (from a different plant species) CENH3 tail. For example, it has been found that GFP fused to a maize CENH3 tail domain linked to an Arabidopsis CENH3 histone-fold domain is active.

As noted above, in some embodiments, the tail domain of an H3 histone (not to be confused with a CENH3 histone) is used as the tail domain portion of the mutated CENH3 protein (these embodiments are sometimes referred to as “tailswap” proteins). Plant H3 tail domains are well conserved in various organisms. For example, a common H3 tail domain from plants is SEQ ID NO:95. Thus, in some embodiments, the heterologous tail portion of the tailswap protein will comprise an amino acid sequence substantially identical (e.g., at least 70, 80, 90, 95, or 100% identical) to SEQ ID NO:95, or a fragment thereof at least 15, 20, 25, 30, 35, or 40 amino acids long.

In some embodiments, the mutated CENH3 proteins of the invention will lack at least a portion (e.g., at least 5, 10, 15, 20, 25, 30, or more amino acids) of the endogenous CENH3 N-terminal region, and thus, in some embodiments, will have a truncated CENH3 tail domain compared to a wildtype endogenous CENH3 protein. Mutated CENH3 proteins may, or may not, be linked to a heterologous sequence.

Optionally, the heterologous amino acid sequence can comprise, or further comprise, one or more amino acid sequences at the amino and/or carboxyl terminus and/or linking the tail and histone fold domains. For example, in some embodiments, the mutated CENH3 protein (e.g., a tailswap or other CENH3 mutated protein) comprises a heterologous amino acid sequence linked to the amino end of the tail domain. In some embodiments, the heterologous sequence is linked to the amino terminus of an otherwise wildtype CENH3 protein, wherein the heterologous sequence interferes with centromere function. For example, it has been found, for example, that green fluorescent protein, when linked to wildtype CENH3, sufficiently disrupts centromeres to allow for production of haploid progeny. It is believed that the heterologous sequence can be any sequence that disrupts the CENH3 protein's ability to maintain centromere function. Thus, in some embodiments, the heterologous sequence comprises a an amino acid sequence of at least 5, 10, 15, 20, 25, 30, 50, or more kD.

In some embodiments, the mutated CENH3 protein will comprise a protein domain that acts as a detectable or selectable marker. For example, an exemplary selectable marker protein is fluorescent or an antibiotic or herbicide resistance gene product. Selectable or detectable protein domains are useful for monitoring the presence or absence of the mutated CENH3 protein in an organism.

B. Non-CENH3 Kinetochore Proteins

It is believed that other proteins that make up the kinetochore complex can also be mutated and expressed in a plant that otherwise does not express the corresponding endogenous kinetochore complex protein to result in a viable plant which, when crossed to a wildtype plant having a wildtype kinetochore complex, generates haploid progeny at a certain frequency (e.g., at least 0.1, 0.5, 1, 5, 10, 20,%, or more). Exemplary non-CENH3 members of the kinetochore complex include, e.g., CENPC, MCM21, MIS12, NDC80, and NUF2.

Active mutated non-CENH3 kinetochore complex proteins (e.g., CENPC, MCM21, MIS12, NDC80, or NUF2) can be identified, for example, by random mutagenesis, single or multiple amino acid targeted mutagenesis, by generation or complete or partial protein domain deletions, by fusion with heterologous amino acid sequences, or combinations thereof. “Active” mutant non-CENH3 kinetochore complex proteins refer to proteins, which when expressed in a plant in which the corresponding non-CENH3 kinetochore complex protein is knocked out or inactivated, results in viable plants, which when crossed to a wildtype plant, produce haploid progeny at a more than normal frequency (e.g., at least 1, 5, 10, 20% or more). In some embodiments, active mutated CENPC, MCM21, MIS12, NDC80, or NUF2 polypeptides are substantially identical to SEQ ID NOs: 96, 97, 98, 99, or 100, respectively. Mutated non-CENH3 kinetochore complex proteins (e.g., CENPC, MCM21, MIS12, NDC80, or NUF2) can be readily tested by recombinant expression of the mutated non-CENH3 kinetochore complex protein in a plant lacking endogenous non-CENH3 kinetochore complex protein, crossing the transgenic plant (as a male or female, depending on fertility) to a plant expressing a wildtype non-CENH3 kinetochore complex protein, and then screening for the production of haploid progeny.

In some embodiments, the mutated non-CENH3 kinetochore complex protein is identical to an endogenous non-CENH3 kinetochore complex protein but for 1, 2, 3, 4, 5, 6, 7, 8, or more (e.g., 1-2, 1-4, 1-7) amino acids. For example, in some embodiments, the endogenous wildtype protein from the plant is identical or substantially identical to any of SEQ ID NOs: 96, 97, 98, 99, or 100 and the mutated non-CENH3 kinetochore complex protein differs from the endogenous non-CENH3 kinetochore complex protein by 1, 2, 3, 4, 5, 6, 7, 8, or more (e.g., 1-2, 1-4, 1-7) amino acids.

Optionally, the heterologous amino acid sequence can comprise one or more amino acid sequences at the amino and/or carboxyl terminus and/or linking the tail and histone fold domains. For example, in some embodiments, the mutated non-CENH3 kinetochore complex protein comprises a heterologous amino acid sequence linked to an amino end of the non-CENH3 kinetochore complex protein. The heterologous sequence can be any sequence. In some embodiments, the heterologous sequence is linked to the amino terminus of an otherwise wildtype non-CENH3 kinetochore complex protein, wherein the heterologous sequence interferes with centromere function. In some embodiments, the heterologous sequence comprises a an amino acid sequence of at least 5, 10, 15, 20, 25, 30, 50, or more kD.

In some embodiments, the mutated non-CENH3 kinetochore complex protein will comprise a protein domain that acts as a detectable or selectable marker. For example, an exemplary selectable marker protein is fluorescent or an antibiotic or herbicide resistance gene product. Selectable or detectable protein domains are useful for monitoring the presence or absence of the mutated non-CENH3 kinetochore complex protein in an organism.

III. Generation of Organisms of the Invention

The present invention provides for organisms that do not express, or express at reduced levels (e.g., less than 90, 80, 70, 60, 50, 40, 30, 20, or 10% of wildtype levels), an endogenous CENH3 protein or non-CENH3 kinetochore complex protein and optionally that express a corresponding mutated CENH3 protein or non-CENH3 kinetochore complex protein. Generally, lack of a kinetochore complex protein is lethal, unless at least partially complemented by a mutated kinetochore complex protein as described herein. Without intending to limit the scope of the invention, it is believed that there are several ways to make an organism that lacks, or has reduced expression of, an endogenous kinetochore complex protein but that expresses a mutated version of that protein.

In some embodiments, one can generate a CENH3 mutation in an endogenous CENH3 (or non-CENH3 kinetochore complex protein) gene that reduces or eliminates CENH3 activity or expression, or generate a kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) gene knockout. In these embodiments, one can generate an organism heterozygous for the gene knockout or mutation and introduce an expression cassette for expression of the heterologous corresponding mutated kinetochore complex protein into the organism. Progeny from the heterozygote can then be selected that are homozygous for the mutation or knockout but that comprise the recombinantly expressed heterologous mutated kinetochore complex protein. Accordingly, the invention provides plants, plant cells or other organisms in which one or both CENH3 alleles are knocked out or mutated to significantly or essentially completely lack CENH3 activity, i.e., sufficient to induce embryo lethality without a complementary expression of a mutated kinetochore complex protein as described herein (e.g., a tailswap protein). The invention also provides plants, plant cells or other organisms in which one or both alleles of a non-CENH3 kinetochore complex gene are knocked out or mutated to significantly or essentially completely lack the corresponding non-CENH3 kinetochore complex protein activity, i.e., sufficient to induce embryo lethality without a complementary expression of a mutated kinetochore complex protein as described herein. In plants having more than a diploid set of chromosomes (e.g. tetraploids), all alleles can be inactivated, mutated, or knocked out.

Alternatively, one can introduce the expression cassette encoding a mutated kinetochore complex protein (e.g., including but not limited to, a tailswap protein) into an organism with an intact set of kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) alleles and then silence the endogenous kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) gene by any way known in the art. As an example, an siRNA or microRNA can be introduced or expressed in the organism that reduces or eliminates expression of the endogenous kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) protein.

Ideally, the silencing siRNA or other silencing agent is selected to silence the endogenous kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) gene but does not substantially interfere with expression of the mutated kinetochore complex protein (e.g., a tailswap protein). In situations where endogenous CENH3 is to be inactivated, this can be achieved, for example, by targeting the siRNA to the N-terminal tail coding section, or untranslated portions, or the CENH3 mRNA, depending on the structure of the mutated kinetochore complex protein. Alternatively, the mutated kinetochore complex protein transgene can be designed with novel codon usage, such that it lacks sequence homology with the endogenous kinetochore complex protein gene and with the silencing siRNA.

IV. Reduction or Elimination of Endogenous Kinetochore Complex Protein Expression

A number of methods can be used to inhibit, mutate, or inactivate expression of a kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) in plants. For instance, antisense technology can be conveniently used to inactivate gene expression. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the antisense strand of RNA will be transcribed. The expression cassette is then transformed into plants and the antisense strand of RNA is produced. In plant cells, it has been suggested that antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the polypeptide of interest, see, e.g., Sheehy et al., Proc. Nat. Acad. Sci. USA, 85:8805-8809 (1988); Pnueli et al., The Plant Cell 6:175-186 (1994); and Hiatt et al., U.S. Pat. No. 4,801,340.

The antisense nucleic acid sequence transformed into plants will be substantially identical to at least a portion of the endogenous gene or genes to be repressed. The sequence, however, does not have to be perfectly identical to inhibit expression. Thus, an antisense or sense nucleic acid molecule encoding only a portion of a kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2), or a portion of the kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) mRNA (including but not limited to untranslated portions of the mRNA) can be useful for producing a plant in which kinetochore complex protein expression is suppressed. The vectors of the present invention are optionally designed such that the inhibitory effect applies only to a kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) and does not affect expression of other genes. In situations where endogenous CENH3 is to be inactivated, one method for achieving this goal is to target the antisense sequence to CENH3 sequences (e.g., tail or untranslated mRNA sequences) not found in other proteins within a family of genes exhibiting homology or substantial homology to the CENH3 gene.

For antisense suppression, the introduced sequence also need not be full length relative to either the primary transcription product or fully processed mRNA. Generally, higher homology can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments may be equally effective. For example, a sequence of between about 30 or 40 nucleotides can be used, and in some embodiments, about full length nucleotides should be used, though a sequence of at least about 20, 50, 100, 200, or 500 nucleotides can be used.

Catalytic RNA molecules or ribozymes can also be used to inhibit expression of a kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs.

A number of classes of ribozymes have been identified. One class of ribozymes is derived from a number of small circular RNAs that are capable of self-cleavage and replication in plants. The RNAs replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples include RNAs from avocado sunblotch viroid and the satellite RNAs from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle virus, solanum nodiflorum mottle virus and subterranean clover mottle virus. The design and use of target RNA-specific ribozymes is described in Haseloff et al. Nature, 334:585-591 (1988).

Another method of suppression is sense suppression (also known as co-suppression). Introduction of expression cassettes in which a nucleic acid is configured in the sense orientation with respect to the promoter has been shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes see, Napoli et al., The Plant Cell 2:279-289 (1990); Flavell, Proc. Natl. Acad. Sci., USA 91:3490-3496 (1994); Kooter and Mol, Current Opin. Biol. 4:166-171 (1993); and U.S. Pat. Nos. 5,034,323, 5,231,020, and 5,283,184.

Generally, where inhibition of expression is desired, some transcription of the introduced sequence occurs. The effect may occur where the introduced sequence contains no coding sequence per se, but only intron or untranslated sequences homologous to sequences present in the primary transcript of the endogenous sequence. The introduced sequence generally will be substantially identical to the endogenous sequence intended to be repressed. This minimal identity will typically be greater than about 65% to the target a kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) sequence, but a higher identity can exert a more effective repression of expression of the endogenous sequences. In some embodiments, sequences with substantially greater identity are used, e.g., at least about 80, at least about 95%, or 100% identity are used. As with antisense regulation, the effect can be designed and tested so as to not significantly affect expression of other proteins within a similar family of genes exhibiting homology or substantial homology.

For sense suppression, the introduced sequence in the expression cassette, needing less than absolute identity, also need not be full length, relative to either the primary transcription product or fully processed mRNA. This may be preferred to avoid concurrent production of some plants that are overexpressers. A higher identity in a shorter than full length sequence compensates for a longer, less identical sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and identity of non-coding segments will be equally effective. In some embodiments, a sequence of the size ranges noted above for antisense regulation is used, i.e., 30-40, or at least about 20, 50, 100, 200, 500 or more nucleotides.

Endogenous gene expression may also be suppressed by means of RNA interference (RNAi) (and indeed co-suppression can be considered a type of RNAi), which uses a double-stranded RNA having a sequence identical or similar to the sequence of the target gene. RNAi is the phenomenon in which when a double-stranded RNA having a sequence identical or similar to that of the target gene is introduced into a cell, the expressions of both the inserted exogenous gene and target endogenous gene are suppressed. The double-stranded RNA may be formed from two separate complementary RNAs or may be a single RNA with internally complementary sequences that form a double-stranded RNA. Although complete details of the mechanism of RNAi are still unknown, it is considered that the introduced double-stranded RNA is initially cleaved into small fragments, which then serve as indexes of the target gene in some manner, thereby degrading the target gene. RNAi is known to be also effective in plants (see, e.g., Chuang, C. F. & Meyerowitz, E. M., Proc. Natl. Acad. Sci. USA 97: 4985 (2000); Waterhouse et al., Proc. Natl. Acad. Sci. USA 95:13959-13964 (1998); Tabara et al. Science 282:430-431 (1998); Matthew, Comp Funct. Genom. 5: 240-244 (2004); Lu, et al., Nucleic Acids Research 32(21):e171 (2004)). For example, to achieve suppression of the expression of a kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) using RNAi, a double-stranded RNA having the sequence of an mRNA encoding the kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2), or a substantially similar sequence thereof (including those engineered not to translate the protein) or fragment thereof, is introduced into a plant or other organism of interest. The resulting plants/organisms can then be screened for a phenotype associated with the target protein (optionally in the presence of expression of a tailswap protein to avoid lethality) and/or by monitoring steady-state RNA levels for transcripts encoding the protein. Although the genes used for RNAi need not be completely identical to the target gene, they may be at least 70%, 80%, 90%, 95% or more identical to the target (e.g., CENH3 sequences as described herein) gene sequence. See, e.g., U.S., Patent Publication No. 2004/0029283 for an example of a non-identical siRNA sequence used to suppress gene expression. The constructs encoding an RNA molecule with a stem-loop structure that is unrelated to the target gene and that is positioned distally to a sequence specific for the gene of interest may also be used to inhibit target gene expression. See, e.g., U.S. Patent Publication No. 2003/0221211.

The RNAi polynucleotides can encompass the full-length target RNA or may correspond to a fragment of the target RNA. In some cases, the fragment will have fewer than 100, 200, 300, 400, 500 600, 700, 800, 900 or 1,000 nucleotides corresponding to the target sequence. In addition, in some embodiments, these fragments are at least, e.g., 10, 15, 20, 50, 100, 150, 200, or more nucleotides in length. In some cases, fragments for use in RNAi will be at least substantially similar to regions of a target protein that do not occur in other proteins in the organism or may be selected to have as little similarity to other organism transcripts as possible, e.g., selected by comparison to sequences in analyzing publicly-available sequence databases.

Expression vectors that continually express siRNA in transiently- and stably-transfected have been engineered to express small hairpin RNAs, which get processed in vivo into siRNAs molecules capable of carrying out gene-specific silencing (Brummelkamp et al., Science 296:550-553 (2002), and Paddison, et al., Genes & Dev. 16:948-958 (2002)). Post-transcriptional gene silencing by double-stranded RNA is discussed in further detail by Hammond et al. Nature Rev Gen 2: 110-119 (2001), Fire et al. Nature 391: 806-811 (1998) and Timmons and Fire Nature 395: 854 (1998).

One of skill in the art will recognize that sense (including but not limited to siRNA) or antisense transcript should be targeted to sequences with the most variance between family members where the goal is to target only one (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) histone family member.

Yet another way to suppress expression of an endogenous plant gene is by recombinant expression of a microRNA that suppresses a target (e.g., a CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2 gene). Artificial microRNAs are single-stranded RNAs (e.g., between 18-25 mers, generally 21 mers), that are not normally found in plants and that are processed from endogenous miRNA precursors. Their sequences are designed according to the determinants of plant miRNA target selection, such that the artificial microRNA specifically silences its intended target gene(s) and are generally described in Schwab et al, The Plant Cell 18:1121-1133 (2006) as well as the internet-based methods of designing such microRNAs as described therein. See also, US Patent Publication No. 2008/0313773.

Methods for introducing genetic mutations into plant genes and selecting plants with desired traits are well known and can be used to introduce mutations or to knock out a kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2). For instance, seeds or other plant material can be treated with a mutagenic insertional polynucleotide (e.g., transposon, T-DNA, etc.) or chemical substance, according to standard techniques. Such chemical substances include, but are not limited to, the following: diethyl sulfate, ethylene imine, ethyl methanesulfonate and N-nitroso-N-ethylurea. Alternatively, ionizing radiation from sources such as, X-rays or gamma rays can be used. Plants having mutated a kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) can then be identified, for example, by phenotype or by molecular techniques.

Modified protein chains can also be readily designed utilizing various recombinant DNA techniques well known to those skilled in the art and described for instance, in Sambrook et al., supra. Hydroxylamine can also be used to introduce single base mutations into the coding region of the gene (Sikorski et al., Meth. Enzymol., 194:302-318 (1991)). For example, the chains can vary from the naturally occurring sequence at the primary structure level by amino acid substitutions, additions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain.

Alternatively, homologous recombination can be used to induce targeted gene modifications or knockouts by specifically targeting the a kinetochore complex protein gene (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) gene in vivo (see, generally, Grewal and Klar, Genetics, 146:1221-1238 (1997) and Xu et al., Genes Dev., 10:2411-2422 (1996)). Homologous recombination has been demonstrated in plants (Puchta et al., Experientia, 50:277-284 (1994); Swoboda et al., EMBO J., 13:484-489 (1994); Offring a et al., Proc. Natl. Acad. Sci. USA, 90:7346-7350 (1993); and Kempin et al., Nature, 389:802-803 (1997)).

In applying homologous recombination technology to the genes of the invention, mutations in selected portions of an kinetochore complex protein gene sequences (including 5′ upstream, 3′ downstream, and intragenic regions) such as those disclosed here are made in vitro and then introduced into the desired plant using standard techniques. Since the efficiency of homologous recombination is known to be dependent on the vectors used, use of dicistronic gene targeting vectors as described by Mountford et al., Proc. Natl. Acad. Sci. USA, 91:4303-4307 (1994); and Vaulont et al., Transgenic Res., 4:247-255 (1995) are conveniently used to increase the efficiency of selecting for altered CENH3 gene expression in transgenic plants. The mutated gene will interact with the target wild-type gene in such a way that homologous recombination and targeted replacement of the wild-type gene will occur in transgenic plant cells, resulting in suppression of kinetochore complex protein activity.

V. Preparation of Recombinant Vectors

To use isolated sequences in the above techniques, recombinant DNA vectors suitable for transformation of plant cells are prepared. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature, e.g., Weising et al., Ann. Rev. Genet. 22:421-477 (1988). A DNA sequence coding for the desired polypeptide, for example the tailswap protein fusions as described herein and/or siRNA, antisense, or other silencing constructs, will be combined with transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the gene in the intended tissues of the transformed plant.

For example, a plant promoter fragment may be employed which will direct expression of the gene in all tissues of a regenerated plant. Alternatively, the plant promoter may direct expression of the polynucleotide of the invention in a specific tissue (tissue-specific promoters), organ (organ-specific promoters) or may be otherwise under more precise environmental control (inducible promoters). Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues, such as fruit, seeds, flowers, pistils, or anthers. Suitable promoters include those from genes encoding storage proteins or the lipid body membrane protein, oleosin.

If proper polypeptide expression is desired, a polyadenylation region at the 3′-end of the coding region should be included. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.

The vector comprising the sequences (e.g., promoters or coding regions) from genes of the invention can also comprise, for example, a marker gene that confers a selectable phenotype on plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron or Basta.

Constitutive Promoters

A promoter, or an active fragment thereof, can be employed which will direct expression of a nucleic acid encoding a fusion protein of the invention, in all transformed cells or tissues, e.g. as those of a regenerated plant. Such promoters are referred to herein as “constitutive” promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include those from viruses which infect plants, such as the cauliflower mosaic virus (CaMV) 35S transcription initiation region (see, e.g., Dagless, Arch. Virol. 142:183-191 (1997)); the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens (see, e.g., Mengiste supra (1997); O'Grady, Plant Mol. Biol. 29:99-108) (1995)); the promoter of the tobacco mosaic virus; the promoter of Figwort mosaic virus (see, e.g., Maiti, Transgenic Res. 6:143-156) (1997)); actin promoters, such as the Arabidopsis actin gene promoter (see, e.g., Huang, Plant Mol. Biol. 33:125-139 (1997)); alcohol dehydrogenase (Adh) gene promoters (see, e.g., Millar, Plant Mol. Biol. 31:897-904 (1996)); ACT11 from Arabidopsis (Huang et al., Plant Mol. Biol. 33:125-139 (1996)), Cat3 from Arabidopsis (GenBank No. U43147, Zhong et al., Mol. Gen. Genet. 251:196-203 (1996)), the gene encoding stearoyl-acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe et al., Plant Physiol. 104:1167-1176 (1994)), GPc1 from maize (GenBank No. X15596, Martinez et al., J. Mol. Biol. 208:551-565 (1989)), Gpc2 from maize (GenBank No. U45855, Manjunath et al., Plant Mol. Biol. 33:97-112 (1997)), other transcription initiation regions from various plant genes known to those of skill. See also Holtorf, “Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana,” Plant Mol. Biol. 29:637-646 (1995). Additional constitutive promoters include, e.g., the polyubiquitin gene promoters from Arabidopsis thaliana, UBQ3 and UBQ10, (Norris et al., Plant Mol. Biol. 21:895 (1993)), are also useful for directing gene expression.

Inducible Promoters

One can optionally use an inducible promoter to control (1) expression of an artificial micro RNA, siRNA, or other silencing polynucleotide, (2) and simultaneously turn on expression of the transgenic mutated (e.g., tailswap) protein, or (3) both (1) and (2). This would have the advantage of having a normal plant (e.g. one that might have higher fertility) until induction, which would then create gametes ready for inducing haploids.

Tissue-Specific Promoters

An alternative is to down-regulate the endogenous protein (e.g. by gene silencing) in a specific tissue (e.g., at least in the mature gametophytes (either pollen or embryo sac)) and to replace it only in this tissue with a specific promoter that drives expression of a tailswap protein. In some embodiments, the same tissue-specific promoter is used to drive an artificial micro RNA, siRNA, or other silencing polynucleotide and the rescuing tailswap-encoding transgene.

VI. Production of Transgenic Plants or Plant Cells

DNA constructs of the invention may be introduced into the genome of the desired plant host by a variety of conventional techniques. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced directly to plant tissue using biolistic methods, such as DNA particle bombardment. Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria.

Microinjection techniques are known in the art and well described in the scientific and patent literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al., Embo J. 3:2717-2722 (1984). Electroporation techniques are described in Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824 (1985). Biolistic transformation techniques are described in Klein et al., Nature 327:70-73 (1987).

Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horsch et al., Science 233:496-498 (1984), and Fraley et al., Proc. Natl. Acad. Sci. USA 80:4803 (1983).

Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype such as increased disease resistance compared to a control plant that was not transformed or transformed with an empty vector. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al., Ann. Rev. of Plant Phys. 38:467-486 (1987).

The nucleic acids and encoded polypeptides of the invention can be used to confer the characteristics described herein, including the ability to generate haploid progeny, as described herein, on essentially any plant. Thus, the invention has use over a broad range of plants, including dicots or monocots, including e.g., species from the genera Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Oryza, Panicum, Pennisetum, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna, and, Zea.

VII. Methods of Improved Breeding

Crossing plants that lack an endogenous kinetochore complex protein and express an active mutated kinetochore complex protein as described herein (e.g., a tailswap or other mutated CENH3 or non-CENH3 kinetochore complex protein) either as a pollen or ovule parent to a plant that expresses an endogenous kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2 protein) will result in at least some progeny (e.g., at least 0.1%, 0.5%, 1%, 5%, 10%, 20% or more) that are haploid and comprise only chromosomes from the plant that expresses the kinetochore complex protein. Thus, the present invention allows for the generation of haploid plants having all of its chromosomes from a plant of interest by crossing the plant of interest with a plant transgenically expressing the mutated kinetochore complex protein and collecting the resulting haploid seed.

As noted above, the plant expressing an endogenous wildtype CENH3 protein can be crossed as either the male or female parent. One unique aspect of the present invention is that it allows for generation of a plant (or other organism) having only a male parent's nuclear chromosomes and a female parent's cytoplasm with associated mitochondria and plastids, when the tailswap parent is the male parent.

While plants lacking an endogenous CENH3 gene and expressing a mutated CENH3 protein made up of GFP-histone H3 tail-CENH3 histone-fold domain have limited male fertility, it has been found that plants lacking an endogenous CENH3 gene and expressing both a mutated CENH3 protein made up of GFP-histone H3 tail-CENH3 histone-fold domain and GFP-wildtype CENH3 results in plants with higher male fertility making them convenient for use as a male, as well as female, parent in crossing. In general, the invention provides for expression of two or more different mutated kinetochore complex proteins in a plant (e.g., a plant lacking expression of the corresponding endogenous kinetochore complex protein(s).

Once generated, haploid plants can be used for a variety of useful endeavors, including but not limited to the generation of doubled haploid plants, which comprise an exact duplicate copy of chromosomes. Such doubled haploid plants are of particular use to speed plant breeding, for example. A wide variety of methods are known for generating doubled haploid organisms from haploid organisms.

Somatic haploid cells, haploid embryos, haploid seeds, or haploid plants produced from haploid seeds can be treated with a chromosome doubling agent. Homozygous double haploid plants can be regenerated from haploid cells by contacting the haploid cells, including but not limited to haploid callus, with chromosome doubling agents, such as colchicine, anti-microtubule herbicides, or nitrous oxide to create homozygous doubled haploid cells.

Methods of chromosome doubling are disclosed in, for example, U.S. Pat. Nos. 5,770,788; 7,135,615, and U.S. Patent Publication No. 2004/0210959 and 2005/0289673; Antoine-Michard, S. et al., Plant Cell, Tissue Organ Cult., Cordrecht, the Netherlands, Kluwer Academic Publishers 48(3):203-207 (1997); Kato, A., Maize Genetics Cooperation Newsletter 1997, 36-37; and Wan, Y. et al., Trends Genetics 77: 889-892 (1989). Wan, Y. et al., Trends Genetics 81: 205-211 (1991), the disclosures of which are incorporated herein by reference. Methods can involve, for example, contacting the haploid cell with nitrous oxide, anti-microtubule herbicides, or colchicine. Optionally, the haploids can be transformed with a heterologous gene of interest, if desired.

Double haploid plants can be further crossed to other plants to generate F1, F2, or subsequent generations of plants with desired traits.

VIII. Non-Plant Organisms

It is believed that the invention is also functional in non-plant organisms that do not have unmatched sex chromosomes. Those of skill in the art can thus generate a mutated kinetochore complex protein (including but not limited to a tailswap protein) based on a particular organism's kinetochore complex protein (e.g., CENH3, CENPC, MCM21, MIS12, NDC80, or NUF2) protein sequence and knockout the corresponding endogenous kinetochore complex protein gene as appropriate for that organisms. Exemplary non-plant organisms for which the invention is believed to be applicable include, but is not limited to, yeast and other fungi, as well as to animals that lack unmatched (e.g., XY) sex or other chromosomes for whom haploids are not viable.

EXAMPLES

The following examples are offered to illustrate, but not to limit the claimed invention.

Production of haploid plants that inherit chromosomes from only one parent can greatly accelerate plant breeding (Dunwell, J. M., Plant Biotechnol J in press; Forster, B. P. et al., Trends Plant Sci 12:368-75 (2007); Forster, B. P. & Thomas, W. T. B. in Plant Breeding Reviews (ed. Janick, J.) 57-88 (John Wiley & Sons, Inc., 2005)). Haploids generated from a heterozygous individual and converted to diploid create instant homozygous lines, bypassing generations of inbreeding. Two methods are generally used to produce haploids: First, cultured gametophyte cells may be regenerated into haploid plants (Guha, S. & Maheshwari, S. C., Nature 204:497 (1964)), but many species and genotypes are recalcitrant to this process (Forster, B. P. et al., Trends Plant Sci 12:368-75 (2007); Wedzony, M. et al. in Advances in Haploid Production in Higher Plants (eds. Touraev, A., Forster, B. P. & Jain, S. M.) 1-33 (Springer, 2009)). Second, haploids can be induced from rare interspecific crosses, in which one parental genome is eliminated after fertilization (Bains, G. S. & Howard, H. W., Nature 166:795 (1950); Barclay, I. R., Nature 256:410-411 (1975); Burk, L. G. et al., Science 206:585 (1979); Clausen, R. E. & Mann, M. C., Proc Natl Acad Sci USA 10:121-124 (1924); Hougas, H. W. & Peloquin, S. J., Nature 180:1209-1210 (1957); Kasha, K. J. & Kao, K. N., Nature 225:874-6 (1970)). The molecular basis for genome elimination is not understood, but one theory posits that centromeres from the two parent species interact unequally with the mitotic spindle, causing selective chromosome loss (Bennett, M. D. et al., Chromosoma 54:175-200 (1976); Finch, R. A., Chromosoma 88:386-393 (1983); Laurie, D. A. & Bennett, M. D., Genome 32:953-961 (1989)). Here it is shown that haploid Arabidopsis thaliana can be easily generated through seeds by manipulating a single centromere protein, the centromere-specific histone CENH3/CENP-A. When cenh3 null mutants expressing altered CENH3 proteins are crossed to wild type, chromosomes from the mutant are eliminated, producing haploid progeny. Haploids are spontaneously converted into fertile diploids through meiotic non-reduction, allowing their genotype to be perpetuated. Maternal and paternal haploids can be generated through reciprocal crosses. Centromere-mediated genome elimination has also been exploited to convert a natural tetraploid Arabidopsis into a diploid, reducing its ploidy to simplify breeding. As CENH3 is universal in eukaryotes, our method can be extended to produce haploids in any plant species.

Centromeres are the chromosomal loci that attach to spindle microtubules to mediate faithful inheritance of the genome during cell division. They are epigenetically specified by incorporation of CENH3 (CENP-A in humans, HTR12 in A. thaliana (Talbert, P. B. et al., Plant Cell 14:1053-66 (2002))), a histone H3 variant that replaces conventional H3 in centromeric nucleosomes (Henikoff, S. & Dalal, Y., Curr Opin Genet Dev 15:177-84 (2005))). Cenh3-1, an embryo-lethal null mutant in A. thaliana that allows us to completely replace native CENH3 with modified variants, was isolated. cenh3-1 plants complemented by transgenic green fluorescent protein-tagged CENH3 (GFP-CENH3) have a wild-type phenotype. cenh3-1 can also be rescued by “GFP-tailswap”, a transgene in which the hypervariable N-terminal tail domain of CENH3 was replaced with the tail of conventional H3, using the H3.3 variant (encoded by At1g13370). GFP-tailswap was tagged at its N-terminus with GF and contained the N-terminal tail of H3 fused to the histone fold domain of CENH3 as follows:

H3 tail: MARTKQSARKSHGGKAPTKQLATKAARKSAPTTGGVKKPHRFR (SEQ ID NO:95) joined to the CENH3 histone fold domain:

(SEQ ID NO: 109) PGTVALKEIRHFQKQTNLLIPAASFIREVRSITHMLAPPQINRWTAEA LVALQEAAEDYLVGLFSDSMLCAIHARRVTLMRKDFELARRLGGKGRP W. “GFP-tailswap” plants (cenh3-1 rescued by a GFP-tailswap transgene) showed accurate mitosis, as aneuploidy in somatic cells was not detected. However, GFP-tailswap plants were sterile upon flowering, indicating that they may have a specific defect in meiosis. GFP-tailswap was mostly male sterile, although it could be used as a pollen donor if many anthers were pooled. When crossed as the female to a wild type male, GFP-tailswap plants were 60-70% as fertile as wild type.

When GFP-tailswap was pollinated by wild type, several unusual phenotypes in F1 progeny were observed. First, 80-95% of fertilized ovules aborted early in development, yielding inviable seeds (Table 1).

TABLE 1 Haploid plants contain only the nuclear genome of their wild type parent. % total nor- plants hap- dip- aneu- seeds/ mal ana- loids loids ploids Cross silique seed lyzed (%) (%) (%) WT Col-0 × 52 ± 6 99.5 224  0 224  0 WT Col-0 (n = 23) (0)  (100) (0)  GFP-tailswap × 0.6 80 213  0 197 16 GFP-tailswap (n = 1206) (0)  (92)  (8)  GFP-tailswap × 32 ± 9 12 67 23  23 21 WT Col-0 (n = 40) (34) (34)  (32) WT Col-0 × nd nd 116  5  99 12 GFP-tailswap (4)  (85)  (11) GFP-tailswap × 30 ± 4 23 127 32*  32 63 WT Ler (n = 22) (25) (25)  (50) GFP-tailswap × 23 ± 5 8 22 10*  7  5 WT Ws-0 (n = 14) (45) (32)  (28) GFP-tailswap × 28 ± 5 30 117 34†  39 44 WT C24/Ler (n = 13) (29) (33)  (38) C24/Ler male- 22 ± 14 63 226 12† 206  8 sterile × (n = 18) (5)  (91)  (4)  GFP-tailswap GFP-CENH3 × 53 ± 4 99 209  0 209  0 GFP-CENH3 (n = 21) (0)  (100) (0)  GFP-CENH3 × 54 ± 7 67 164  8 109 47 WT (n = 18) (5)  (66)  (29) WT × 48 ± 6 96 112  0 108  4 GFP-CENH3 (n = 13) (0)  (96)  (4)  dip- trip- aneu- loids loids ploids (%) (%) (%) GFP-tailswap × 21 ± 6 1.8 41 11  0 30 Wa-1 (n = 96) (27) (73) (tetraploid)

Second, while viable offspring were expected to be diploids heterozygous for cenh3-1 and hemizygous for the GFP-tailswap transgene, 10 out of 16 plants had only wild-type CENH3 and lacked GFP-tailswap. Each of these plants was sterile despite having a wild-type genotype. Furthermore, crossing GFP-tailswap to a quartet mutant male also yielded sterile F1 offspring (3/5 plants) that showed the quartet mutant phenotype of fused pollen, despite the fact that quartet is recessive and the GFP-tailswap parent was expected to transmit a wild-type QUARTET allele. These striking observations suggested that sterile progeny had lost chromosomes from their GFP-tailswap mother, and thus had fewer chromosomes than diploid A. thaliana (2n=10). The karyotype of these plants was examined and found them to be haploids containing only five chromosomes.

As centromeres control chromosome inheritance, it was reasoned that chromosomes that entered the zygote containing the GFP-tailswap variant of CENH3 would be missegregated and lost, creating haploid plants with chromosomes only from their wild type parent. To confirm this, GFP-tailswap plants (in the Col-0 accession) were crossed to several polymorphic accessions and genotyped F1 haploids for markers on all five A. thaliana chromosomes (Table 1). Regardless of the wild-type parent used, haploid plants invariably contained only wild-type chromosomes (paternal haploids), indicating that the GFP-tailswap genome was eliminated (a total of 42 haploids were genotyped). Further, our results show that the process of inducing haploids by centromere-mediated genome elimination is independent of the genotype of the wild-type parent.

Genome elimination induced by CENH3 alterations is not specific to the GFP-tailswap transgene. Crossing cenh3-1 mutants complemented by GFP-CENH3 to wild type also yielded haploid plants, but at a lower frequency than GFP-tailswap (Table 1). Haploid progeny from self-fertilized GFP-tailswap or GFP-CENH3 plants were not observed (Table 1). Our results suggest that general perturbations in centromere structure are sufficient to impede chromosome segregation during zygotic mitosis, creating a haploid embryo when chromosomes containing mutant CENH3 compete with wild type on the same spindle.

Haploids are efficiently generated from a GFP-tailswap×wild type cross, comprising 25-45% of viable offspring (Table 1). Remaining progeny were either diploid hybrids, or aneuploid hybrids showing the developmental phenotypes typical of A. thaliana plants with more than 10 chromosomes (Henry, I. M. et al., Genetics 170:1979-88 (2005)) (Table 1). Aneuploidy might also account for the high level of seed abortion in a GFP-tailswap×wild type cross, as some embryos with unbalanced karyotypes may be inviable.

Uniparental haploids may contain the genome of either their female or male parent. Haploids were also obtained by crossing a wild-type female to GFP-tailswap as the pollen donor (Table 1). In this case, haploid progeny are purely maternal in origin. Genotyping of the plastid genome showed that both maternal and paternal haploids contained the cytoplasm of their maternal parent. Either maternal or paternal haploids were made by using GFP-tailswap plants as the male or female parent respectively in a cross to wild type. The proportion of haploids and aneuploids was much lower if a wild-type female was crossed to a GFP-tailswap male (Table 1). It is hypothesized that if CENH3 is expressed earlier in development from the maternal (wild type) genome, wild-type CENH3 could be incorporated into paternal chromosomes derived from GFP-tailswap, preventing genome elimination in a wild type×GFP-tailswap cross.

Haploid A. thaliana plants are morphologically similar to diploids, but are comparatively smaller in size. Early in vegetative development, haploids have narrower rosette leaves. After bolting, haploids produce more leaves from secondary meristems. Haploid flowers are smaller than diploid flowers, following the general trend that flower size increases with ploidy in A. thaliana. Haploids are generally sterile. They contain a single copy of each chromosome and cannot undergo homologue pairing in meiosis, resulting in gametes that do not contain a full complement of chromosomes. Maternal and paternal haploid plants had similar adult morphology. This is consistent with the fact that all documented imprinting in A. thaliana occurs in the short-lived endosperm, a structure confined to the seed.

To exploit the potential of haploids in crop improvement, their genome should be doubled to generate fertile diploids (doubled haploids) (Forster, B. P., et al. Trends Plant Sci 12:368-75 (2007)). A close inspection of A. thaliana haploids revealed that random siliques had one or two seeds. Each haploid plant yielded a total of 50-2500 seeds depending on the wild-type parental accession (Table S1).

TABLE S1 Number of spontaneous diploid seeds produced by A. thaliana haploids Plant Col-0 Ler Ws-0 1 54 951 1662 2 68 2115 293 3 91 352 2343 4 214 520 1532 5 349 325 2679 6 421 101 215 7 537 219 913 8 121 1013 9 134 424 10  85 630 11  99 1346 Mean 197.5454545 726.9090909 1376.714286 Standard deviation 164.1294389 594.8035734 955.1793399

A majority (95%) of these seeds appeared normal and gave rise to fertile diploids. To address how haploids gave rise to diploid seeds, chromosome segregation during haploid male meiosis was analyzed. During prophase I the five chromosomes remained separate as univalents, which aligned properly in metaphase I. In anaphase I, most meiocytes showed unbalanced reductional segregation (4-1, 3-2, etc.). Meiosis II in these cases gave rise to aneuploid tetrads. In a small minority of anaphase I cells, the 5 univalents migrated towards one pole (5-0 segregation). In subsequent meiosis II, sister chromatids segregated equally, giving rise to haploid dyads and viable gametes. Thus, it is assumed that occasional non-reduction during both male and female haploid meiosis yielded doubled haploids through self-fertilization, consistent with previous observations (Chase, S. S., Botanical Review 35:117-167 (1969); Jauhar, P. P. et al., Crop Science 40:1742-1749 (2000)). In rare instances, spontaneous chromosome doubling in somatic tissues of haploid A. thaliana plants was observed; a side branch from the main inflorescence (2 out of 78 plants) or a random silique (6 out of 78 plants) showed a complete seed set. The microtubule polymerization inhibitor colchicine also induces somatic chromosome doubling in haploid A. thaliana, and diploid shoots that regenerate after treatment show complete seed set. Although A. thaliana haploids have been produced through anther culture (Avetisov, V. A., Genetika, 12:17-25 (1976)), spontaneous diploids recovered in these experiments were reportedly sterile (Scholl, R. & Amos, J. A., Z Pflanzenphysiol 96:407-414 (1980)), and the method has not been widely adopted. The ease of generating haploids through seed by altering CENH3, and of converting haploids into diploids allows large scale generation of doubled haploids in A. thaliana.

Many commercial crops are polyploid (Udall, J. A. & Wendel, J. F., Crop Sci, 46:S3-S14 (2006)), but genetic analysis of polyploids is tedious. Reducing the ploidy of these crops will facilitate easy breeding, so it was tested whether centromere-mediated genome elimination could scale down a tetraploid to diploid. A. thaliana is predominantly diploid, but tetraploid accessions exist (Henry, I. M. et al., Genetics 170:1979-88 (2005)). GFP-tailswap was crossed to the natural tetraploid Warschau-1 (Wa-1), and although over 98% of seed were aborted, viable F1 progeny included synthetic diploid plants containing only Wa-1 chromosomes (Table 1). Therefore, it is possible to extend centromere-mediated genome elimination to halve the ploidy of polyploids.

Centromere incompatibility was previously hypothesized to cause selective genome elimination in interspecies crosses (Bennett, M. D. et al., Chromosoma 54:175-200 (1976); Finch, R. A., Chromosoma 88:386-393 (1983); Laurie, D. A. & Bennett, M. D., Genome 32:953-961 (1989); Heppich, S. et al., Theor Appl Genet 61:101-104 (1982); Jin, W. et al., Plant Cell 16:571-81 (2004)), but it was not known how centromeres could be manipulated to achieve this. It was established a practical basis for engineering genome elimination by altering CENH3, a protein essential for centromere function in all eukaryotes. The fact that haploids were produced with both GFP-tailswap and GFP-CENH3 transgenes suggests that multiple different alterations to the protein may induce genome elimination in other plants. A. thaliana plants that coexpress wild-type and GFP-tailswap or GFP-CENH3 proteins do not act as a haploid inducer. Therefore, our method currently relies on replacing native CENH3 with an altered variant. A cenh3 mutation or a gene silencing method such as RNA interference could be used to reduce or eliminate endogenous CENH3 function in a novel species.

Haploid inducing lines have been described in the grasses (Coe, E. H., American Naturalist 93:381-382 (1959); Hagberg, A. & Hagberg, G., Hereditas 93:341-343 (1980); Kermicle, J. L., Science 166:1422-1424 (1969)), but their genetic basis is not known, except for maize indeterminate gametophyte (ig) (Evans, M. M., Plant Cell 19:46-62 (2007)). The effect of ig may be limited to maize, because mutations in the A. thaliana ig orthologue AS2 do not phenocopy its effect (Ori, N. et al., Development 127:5523-32 (2000)). Our process has key advantages over current methods for producing haploid plants. 1) No tissue culture is needed, removing a major source of genotype dependence. 2) The same inducer produces maternal and paternal haploids. 3) Crossing a cenh3 mutant as the female transfers the nuclear genome of the male parent into a heterologous cytoplasm. This could accelerate production of cytoplasmic male sterile lines for making hybrid seed. 4) Genome elimination occurs between parents that are isogenic except for CENH3 alterations, avoiding fertility barriers inherent to wide crosses.

Genome elimination induced by changes in CENH3 probably occurs during the first few zygotic mitoses, when centromeres from the two parents are loaded with different populations of CENH3 proteins. Expression of both wild-type and mutant CENH3 genes in subsequent cell cycles should rapidly equalize the amount of the two proteins in individual centromeres. Zygotic mitosis is normal in GFP-tailswap and in GFP-CENH3 plants, because haploids from self-fertilized plants were not observed. Furthermore, GFP-CENH3 plants have a completely wild type phenotype. Subtle differences in centromere DNA binding, kinetochore assembly, or coupling to spindle microtubules may be sufficient to slow the segregation of chromosomes containing altered CENH3, resulting in genome elimination. Cell cycle checkpoints in plants must be relaxed enough to allow wild type and mutant chromosomes to segregate differentially, and presumably to permit cytokinesis without complete chromosome segregation. The precise mechanism of genome elimination in our experiments remains unknown.

Centromere DNA sequences and the CENH3 protein both evolve rapidly, and centromere differences have been proposed to create species barriers (Henikoff, S. et al., Science 293:1098-102 (2001)). Although our experiments used tagged proteins, they indicate that changes in CENH3 can induce specific chromosome loss in a hybrid zygote.

Methods Summary

Plant Materials.

cenh3-1 is a G-to-A transition at nucleotide 161 relative to ATG=+1, and mutates a conserved splice acceptor in the second intron. GFP-CENH3 and GFP-tailswap transgenes contained an N-terminal GFP, and used the endogenous CENH3 promoter and terminator. The location of the GFP-tailswap transgene was determined by TAIL-PCR, allowing us to determine whether the transgene was homozygous or hemizygous. The C24/Ler male sterile line was a gift from Dr Luca Comai (University of California, Davis). Male sterility was conferred by the A9-barnase transgene. Plants were grown under 16 hrs of light/8 hours of dark at 20 degrees C.

Genomic DNA Preparation and Genotyping.

Genomic DNA preparation and PCR genotyping were performed using standard methods.

Cytogenetic Analysis.

To analyze meiotic progression and to determine ploidy, mitotic and meiotic chromosome spreads from anthers were prepared according to published protocols.

Plant Materials

cenh3-1 was isolated by the TILLING procedure (Comai, L. & Henikoff, S., Plant J 45:684-94 (2006)). The TILLING population was created by mutagenizing Arabidopsis thaliana in the Col-0 accession with ethylmethanesulfonate, using standard protocols. Cenh3-1 was isolated by TILLING using the CEL1 heteroduplex cleavage assay, with PCR primers specific for the CENH3/HTR12 gene.

cenh3-1 is predicted to disrupt normal splicing of CENH3, because it mutates a conserved splice acceptor site at the beginning of the second coding exon. Translation of an mRNA containing the first coding exon spliced to an incorrect location within CENH3 is predicted to yield only 18 correct amino acids. As the histone-fold domain of CENH3 begins at amino acid residue 82, it is believed that cenh3-1 is a null allele (this is supported by its embryo-lethal phenotype).

Cloning of the GFP-CENH3 and GFP-tailswap transgenes, and construction of the complemented cenh3-1 GFP-CENH3 and cenh3-1 GFP-tailswap lines are described elsewhere (Ravi, Comai, Sundaresan, Chan et al, manuscript in preparation). Primer sequences and full details are available on request.

To cross wild type as the female to GFP-tailswap as the male, a dissecting microscope was used to directly observe pollen deposition on the stigma (GFP-tailswap is mostly male-sterile). The amount of viable pollen in individual flowers of GFP-tailswap varies. Flowers that clearly showed higher amounts of pollen were selected, and pollinated with more than 60 anthers (10 GFP-tailswap flowers) per wild type stigma to achieve the seed set reported in Table 1. Using an optivisor (magnifying lens) and approximately 12 anthers (2 GFP-tailswap flowers) per wild type stigma, a much lower seed set per silique was obtained.

The percentage of normal seeds was determined by visual inspection using a dissecting microscope.

Seed from GFP-tailswap×wild type crosses were sown on 1×MS plates containing 1% sucrose to maximize germination efficiency, particularly of seed that had an abnormal appearance. Late germinating seeds were frequently haploid.

The quartet mutant used was qrt1-2 (Francis, K. E. et al., Plant Physiol 142:1004-13 (2006)).

Male sterility in the C24/Ler line was conferred by the A9-barnase transgene (Bushell, C. et al., Plant Cell 15:1430-42 (2003); Paul, W. et al., Plant Mol Biol 19:611-22 (1992)).

In the GFP-tailswap×Wa-1 experiment, progeny from the GFP-tailswap×Wa-1 cross that contained only Wa-1 chromosomes were confirmed as diploid using chromosome spreads. Plants that were heterozygous for some chromosomes (Col-0 and Wa-1 markers) and homozygous for other chromosomes (Wa-1 markers only) were scored as aneuploid. Triploid offspring (heterozygous for markers on all chromosomes) were not found. A subset of plants were further karyotyped by means of chromosome spreads to confirm aneuploidy.

Cytogenetic Analysis.

Mitotic and meiotic chromosome spreads from anthers were prepared according to published protocols (Ross, K. J. et al., Chromosome Res 4:507-16 (1996)).

Colchicine Treatment

Colchicine treatment of developing haploid plants used a previously published protocol with minor modifications (Josefsson, C. et al., Curr Biol 16:1322-8 (2006)). A solution of 0.25% colchicine, 0.2% Silwet was prepared, and a 20 μL drop was placed on the meristem prior to bolting. Plants became transiently sick after colchicine treatment. Upon recovery, fertile inflorescences appeared from secondary meristems indicating successful chromosome doubling. Haploid plants can also be treated after bolting, although the rate of success is considerably lower.

Example 2 GFP-Maizetailswap Creates F1 Haploids in a Cross to Wild Type

A chimera was created in which the A. thaliana CENH3 tail from CENH3 is replaced with the CENH3 tail domain (SEQ ID NO:102) from maize (Zea mays), thereby generating a fusion of the maize CENH3 tail and A. thaliana CENH3 histone-fold domain, and transformed the fusion into cenh3-1 heterozygotes. As expected, this GFP-maizetailswap protein was targeted to kinetochores and rescued the embryo-lethal phenotype of cenh3-1. Complemented plants were more sterile than GFP-tailswap complemented plants, but had limited fertility when used as the female. When cenh3-1 GFP-maizetailswap females were crossed to wild-type males, 2 haploids, 3 diploids and 5 aneuploids were found among a total of 10 F1 progeny.

mCherry-Tailswap Creates F1 Haploids in a Cross to Wild Type

A transgene was created in which the GFP tag in GFP-tailswap was replaced with an N-terminal mCherry tag (mCherry (SEQ ID NO:105) is a monomeric version of the red fluorescent protein DsRed). From N-terminus to C-terminus, this protein contains mCherry, the tail domain of Arabidopsis thaliana H3.3, and the histone fold domain of Arabidopsis thaliana CENH3. mCherry-tailswap transgenes were transformed into cenh3-1 heterozygotes. When complemented cenh3-1 mCherry-tailswap plants were crossed as female to wild-type male, 1 haploid, 6 aneuploids and 4 diploids were observed from 11 F1 progeny.

The mCherry-tailswap construct was made as a CP 169 vector pCAMBIA 1300 with an HTR promoter. The insert included a Mlu I site followed by the N terminal mcherry Sal I XbaI followed by HTR12 terminator. The H3 Tailswap fragment was synthesized by overlapping PCR and digested with SalI+XbaI and cloned into CP169 to make the mcherrytailswap construct.

A Tailswap Transgene with No GFP Tag Complements a cenh3-1 Mutation. Complemented Plants Create F1 Haploids in a Cross to Wild Type

A transgene was created in which the GFP tag in GFP-tailswap was removed. From N-terminus to C-terminus, this protein contains the tail domain of Arabidopsis thaliana H3.3, and the histone fold domain of Arabidopsis thaliana CENH3. tailswap transgenes were transformed into cenh3-1 heterozygotes. When complemented cenh3-1 tailswap plants were crossed as female to wild-type male, 4 haploids, 27 aneuploidy and 67 diploids were observed from 95 F1 progeny.

Co-Expression of Different CENH3 Variants Creates Desirable Properties in a Genome Elimination Strain.

The previously described GFP-tailswap plant (cenh3-1 mutant plants rescued by a GFP-tailswap transgene) is a very efficient haploid inducer, but is difficult to cross as the pollen donor because it is mostly male sterile. GFP-CENH3 (cenh3-1 mutant plants rescued by a GFP-CENH3 transgene) is a weaker haploid inducer but is much more fertile. It was found that co-expression of GFP-CENH3 and GFP-tailswap in cenh3-1 plants would produce more viable pollen than GFP-tailswap, yet still induce genome elimination when these plants were crossed to wild-type diploid or tetraploids. Indeed, cenh3-1 carrying both GFP-CENH3 and GFP-tailswap transgenes (GEM; Genome Elimination caused by a Mix of cenh3 variants) plants produced ample pollen for crosses, although pollen viability was still lower than wild-type.

Crossing GEM females to wild-type males yielded 2 F1 haploids from 50 progeny. When wild-type females were crossed to GEM males, one haploid was found from 104 progeny.

GEM plants are a major improvement over GFP-tailswap or GFP-CENH3 when the wild-type parent is a tetraploid that has diploid gametes. When GEM plants were crossed as male or female to tetraploid wild-type, chromosomes from the GEM parent were eliminated in a subset of F1 progeny (Table 3). GEM is fertile as either male or female, and shows efficient genome elimination when crossed to a tetraploid parent with diploid gametes.

TABLE 2 Crosses between GEM and diploid wild-type plants produce genome elimination. Total plant Uniparental* cross (♀ × ♂) analysed Triploid Aneuploid diploid plants Wild type 4n × GEM 85 53 27 5 GEM × Wild type 4n 84 12 57 15

TABLE 3 Crosses between GEM and tetraploid wild-type plants produce genome elimination. Total plant Uniparental* cross (♀ × ♂) analysed Diploid Aneuploid haploid plants Wild type 2n × GEM 104 62 18 1 GEM × Wild type 2n 50 36 12 2 Methods for GFP-Maizetailswap Construction

Maize tailswap CENH3 transgene was constructed by fusing in frame the Maize CENH3 N-terminal tail (corresponding to 1-61aa) and Arabidopsis CENH3 histone fold domain (corresponding to 82-179 aa) by overlapping PCR. The maize N terminal tail domain (206 bp) was amplified from maize cDNA using the primer combinations CP 384 (5′-NNNNgtcgacATGGCTCGAACCAAGCACCA-3′ (SEQ ID NO:110), SalI site is italicized) and CP 572 (5′-CAACGGTTCCTGGCCTCCAGCGGTGGC-3′ (SEQ ID NO:111)). The Arabidopsis HFD (950 bp) was amplified from genomic DNA using primer combinations CP 571 (5′-GCCACCGCTGGAGGCCAGGAACCGTTG-3′ (SEQ ID NO:112)) and CP 375 (5′-NNNNtctagaTCACCATGGTCTGCCTTTTCCTCC-3′ (SEQ ID NO:113), XbaI site is italicized). The resultant fragments were gel purified and used as a template to fuse them in an overlapping PCR using primer combinations CP 384 and CP 375. The resultant 1.15 kb fragment is cloned as a SalI-XbaI fragment in a binary vector CP 93 (derived from pCAMBIA 1300). The vector CP 93 contains GFP coding sequence upstream in frame with SalI-XbaI site and its expression is controlled by the 5′ and 3′ regulatory sequences of Arabidopsis CENH3 gene.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. 

What is claimed is:
 1. A transgenic plant comprising a heterologous transgene expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide encoding a polypeptide comprising a heterologous amino acid sequence of at least 5 amino acids linked to an amino terminus of a protein comprising a CENH3 histone-fold domain, wherein the heterologous amino acid sequence is heterologous to the CENH3 histone-fold domain.
 2. The transgenic plant of claim 1, wherein one or two alleles of the endogenous CENH3 genomic coding sequence of the plant is inactivated or knocked out.
 3. The transgenic plant of claim 2, wherein all alleles of the endogenous CENH3, genomic coding sequence of the plant are inactivated or knocked out.
 4. The transgenic plant of claim 3, wherein the plant, when crossed with a wildtype diploid plant, generates at least 0.1% haploid progeny.
 5. The transgenic plant of claim 1, wherein the heterologous amino acid sequence is linked to the CENH3 histone-fold domain via an intervening protein sequence.
 6. The transgenic plant of claim 5, wherein the intervening protein sequence comprises a non-CENH3 histone H3 tail domain.
 7. The transgenic plant of claim 5, wherein the intervening protein sequence comprises a CENH3 tail domain.
 8. The transgenic plant of claim 7, wherein the CENH3 tail domain is heterologous to the CENH3 histone-fold domain.
 9. The transgenic plant of claim 5, wherein the heterologous amino acid sequence is at least 10 amino acids long.
 10. The transgenic plant of claim 1, wherein the heterologous amino acid sequence comprises a non-CENH3 histone H3 tail domain and the non-CENH3 histone H3 tail domain is linked to an amino terminus of a CENH3 histone-fold domain.
 11. An isolated nucleic acid comprising a polynucleotide encoding the polypeptide of claim
 1. 12. A method of generating an F1 progeny plant having half the ploidy of a parent plant expressing an endogenous CENH3 protein, the method comprising, crossing the parent plant to the plant of claim 3; and selecting F1 progeny generated from the crossing step having half the ploidy of the parent plant.
 13. The method of claim 12, wherein the parent plant is the pollen parent.
 14. The method of claim 12, wherein the parent plant is the ovule parent.
 15. The method of claim 12, wherein the parent plant is diploid and the selected F1 progeny is haploid and the method further comprises converting at least one selected haploid plant into a doubled haploid plant.
 16. A method of making the transgenic plant of claim 1, comprising, transforming plant cells with a nucleic acid comprising the expression cassette; and selecting transformants comprising the nucleic acid, thereby making the transgenic plant.
 17. The method of claim 12, wherein the parent plant is a diploid and the selected F1 progeny are haploid.
 18. The transgenic plant of claim 6, wherein the heterologous amino acid sequence comprises green fluorescent protein.
 19. The transgenic plant of claim 7, wherein the heterologous amino acid sequence comprises green fluorescent protein.
 20. The transgenic plant of claim 8, wherein the heterologous amino acid sequence comprises green fluorescent protein.
 21. The isolated nucleic acid of claim 11, wherein the heterologous amino acid sequence is linked to the CENH3 histone-fold domain via an intervening protein sequence.
 22. The method of claim 12, wherein the heterologous amino acid sequence is linked to the CENH3 histone-fold domain via an intervening protein sequence.
 23. The isolated nucleic acid of claim 21, wherein the intervening protein sequence comprises a non-CENH3 histone H3 tail domain.
 24. The method of claim 22, wherein the intervening protein sequence comprises a non-CENH3 histone H3 tail domain.
 25. The isolated nucleic acid of claim 11, wherein the heterologous amino acid sequence comprises a non-CENH3 histone H3 tail domain and the non-CENH3 histone H3tail domain is linked to an amino terminus of a CENH3 histone-fold domain.
 26. The method of claim 12, wherein the heterologous amino acid sequence comprises a non-CENH3 histone H3 tail domain and the non-CENH3 histone H3 tail domain is linked to an amino terminus of a CENH3 histone-fold domain. 